- 學位論文
用即時聚合酶連鎖反應快速偵測乙型鏈球菌於新生兒的應用
Rapid detecting Group B Streptococci using a real-time polymerase chain reaction assay in newborn
摘要
背景:乙型鏈球菌是過去這三十年來早發型新生兒敗血症最主要的致病菌.大約有百分之十到百分之三十的懷孕婦女直腸陰道中有乙型鏈球菌的帶原,而垂直傳播給其寶寶的機率約為百分之五十.美國疾病管制局於2002年發表的準則中建議,所有妊娠35到37週的婦女,都應該檢查生殖道的乙型鏈球菌的帶原狀態,並對帶原者於待產時投予靜脈抗生素治療.因為這項準則的推廣,乙型鏈球菌造成新生兒敗血症之盛行率已經大幅的減少.然而,這準則是基於這種以培養做篩檢方式帶來的保護效應為基礎,但實際上,這種以培養為基礎的篩檢方式仍有它的限制.例如:因為培養結果耗時,產婦在待產時細菌培養報告還未知,或是急產之產婦來不及投予靜脈的抗生素,或是做細菌培養時(妊娠35-37週)與生產時乙型鏈球菌的帶原狀況不同.另外更重要的是,早產兒是感染乙型鏈球菌的高危險族群.但因為早產,往往母親尚未執行乙型鏈球菌的篩檢.有鑒於此,近年來, 以即時聚合酶連鎖反應測試來檢測乙型鏈球菌,開始漸漸被應用在待產的產婦身上. 若以待產時傳統的細菌培養為黃金標準,此即時聚合酶連鎖反應測試比起產前的細菌培養有著更高的敏感性與即時性.然而,目前以聚合酶連鎖反應測試偵測乙型鏈球菌只有應用於待產的婦女身上,尚未直接用於檢測新生兒乙型鏈球菌的帶原狀況. 目的:1.評估以即時聚合酶連鎖反應測試,直接偵測新生兒乙型鏈球菌帶原的準確度.2.評估新生兒呼吸窘迫症與新生兒乙型鏈球菌帶原的相關性 材料與方法:收案對象為凡在2009年6月至2010年5月,於國泰醫院出生,母親具有乙型鏈球菌帶原或是出生後一小時內出現呼吸窘迫,需要住進新生兒加護病房或是觀察室的新生兒.所有收案對象在出生後第一次洗澡前立即以特殊的抹棒採取外耳道,鼻腔,臍帶週遭與腋下皮膚的液體,並同時收集口腔的分泌物或是胃液.所有檢體先以羊血培養皿培養,然後用GBS grouping Latex kit去證實為乙型鏈球菌,在同時,所有收集的検體以DI-Strep B PCR assay (Becton Dickinson & Co., Franklin Lakes, NJ, USA)執行乙型鏈球菌的即時聚合酶連鎖反應.此外,我們也收集寶寶的第一泡尿液去做尿中乙型鏈球菌抗原測試.以乙型鏈球菌培養結果作為黃金標準,比較即時聚合酶連鎖反應的敏感度並與母親及寶寶的週產期資料做比較. 結果:母親在妊娠35-37週直腸生殖道的乙型鏈球菌帶原率為15.4%.85位新生兒是由有乙型鏈球菌帶原的母親所生,其中74位是經由自然產出生,15位(20.2%)的乙型鏈球菌即時聚合酶連鎖反應為陽性,11 位乙型鏈球菌培養為陽性.垂直傳播率為14.9%. 另外11位是經由剖腹產出生, 其中只有一位的乙型鏈球菌即時聚合酶連鎖反應及培養為陽性.所有新生兒的尿液乙型鏈球菌抗原測試結果皆為陰性且無任何一位新生兒發病.比較乙型鏈球菌即時聚合酶連鎖反應呈現陽性與陰性兩組寶寶的週產期資料,發現他們在出生體重,週數,母親年齡與胎次,母親血中白血球數,是否有早期破水…等兩組並無差異.但是乙型鏈球菌即時聚合酶連鎖反應陽性組,明顯的接受待產時靜脈抗生素治療的時間較陰性組短,達到統計顯著意義. 總共有25位新生兒出生後一小時內立即產生呼吸窘迫而收案,其中有六位(24%)的乙型鏈球菌即時聚合酶連鎖反應為陽性.總共有100組配對的乙型鏈球菌即時聚合酶連鎖反應與培養的結果,乙型鏈球菌即時聚合酶連鎖反應的靈敏度為100%(95% CI 71.5~100), 特異度為92% (95% CI 84.5~96.8), 執行所耗的時間約為90分鐘. 結論: 乙型鏈球菌即時聚合酶連鎖反應是偵測新生兒乙型鏈球菌帶原的一項敏感且快速的選擇.
並列摘要
Background: Group B Streptococcus (GBS) infection has been the leading cause of early-onset neonatal sepsis for more than 30 years. Approximately 10-30% of pregnant women are colonized with GBS in their vagina or rectum and the vertical transmission rate was around 50%.The guidelines of CDC 2002 recommend that all pregnant women be screened for GBS carriage between GA 35 and 37 weeks and that intrapartum antibiotic prophylaxis (IAP) be given to carriers. This guideline significantly decreased incidence of GBS related neonatal sepsis. However, there are still some limitations of this culture-based screening strategy, including time consuming and the possibility of changing colonization status after testing in pregnant women. Moreover, infants born preterm have an elevated risk for early onset GBS disease but the colonization status is sometimes unknown at delivery. Recently, a real time polymerase chain reaction (RT-PCR) assay for GBS had been applied to the intrapartum pregnant women with higher sensitivities than the antepartum standard selective broth vaginal-rectal culture. This RT-PCR assay had never been applied directly to newborn in detecting GBS colonization status. Purposes of Study: 1.To evaluate the performance of GBS RT-PCR assay in direct detecting GBS colonization status of newborn. 2. To access the association between neonatal respiratory distress and asymptomatic GBS colonization. Method: Neonates born to a GBS colonization mother or neonates with respiratory distress within 1 hour after birth were enrolled from June, 2009 to May, 2010 in Cathay general hospital. Swabs were obtained from their external ear canals, nostrils, body surface of periumbilical area and subaxillary skin folds immediately after birth before their fist bath. Oral secretion or gastric juice was also collected. All specimens were sent for the IDI-Strep B PCR assay (Becton Dickinson & Co., Franklin Lakes, NJ, USA). Samples were plating and inoculated in the sheep blood agar plate at the same time. Ifβ-hemolytic colony was found on the agar plate, GBS grouping Latex kit was used to reconfirm it. Their urine was collected immediately and the urine latex test was performed to identify the GBS specific antigen. Using GBS culture as the gold standard, we compared the performance of the RT-PCR, urine antigen immunoassay, and correlated with their perinatal characters. Result: The maternal vaginal-rectal GBS colonization rate at gestational age 35-37 weeks was 15.4%. Eighty-five neonates were born to GBS colonization mothers. Among them, 74 were born via vaginal delivery and 15 of them (20.2%) had positive GBS RT-PCR and 11 of them concurrently showed positive GBS culture. The vertical transmission rate of GBS was 14.9%.In the remaining 11 neonates who were born via Cesarean section, only 1 newborn had positive GBS RT-PCR and culture. The urine GBS antigen tests were negative in all enrolled candidates. Comparing neonates with positive or negative of GBS RT-PCR, there wasn’t significant difference in their gestational age, birth body weight, maternal leukocyte count, maternal age but neonates with shorter duration of IAP before delivery had a significant higher GBS PCR positive rate. There were 25 neonates suffering from respiratory distress within 1 hour after birth. Six of them (24%) were GBS PCR positive. A total of 100 paired GBS RT-PCR samples and GBS cultures were available. The sensitivity and specificity of the PCR test were 100% (95% CI 71.5~100) and 92% (95% CI 84.5~96.8), respectively. The time taken to undertake the RT-PCR assay was 90 minutes in average. Conclusion: GBS RT-PCR assay maybe an alternative sensitive method to rapid detection GBS colonization status in newborn.